Immunotherapy in many different forms is making an increasingly strong impact on the treatment success in lymphoid malignancies. Monoclonal antibodies have become a mainstay of treatment. Novel agents, such as the immunomodulatory drugs thalidomide and lenalidomide appear to act primarily through a immune effect. And allogeneic stem cell transplantation is often the only successful salvage treatment for patients with refractory disease. In a collaborative study with the NCI lymphoma team, we have obtained CLL cells from patients who undergo therapy incorporating rituximab. Mechanisms how the monoclonal anti-CD20 antibody rituximab depletes B-cells include antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. In vitro studies have suggested that R induced pro-apoptotic signals contribute to clinical efficacy and may sensitize malignant cells to chemotherapy. To investigate the effect of rituximab on tumor biology in vivo, we characterized the molecular changes in chronic lymphocytic leukemia cells of 12 treatment naive patients during the first rituximab infusion. We identified about 80 genes up-regulated in response to rituximab infusion, many of which are known to be regulated by interferon (IFN) and to have pro-apoptotic function. We found that IFN gamma was consistently upregulated in the serum within the first 6 hours of treatment. Considering the long half-life of the monoclonal antibody, we were surprised to see that both cytokine serum levels and gene expression changes almost completely subsided by 24 hours. We found that CD20 levels were markedly decreased already at 6 hours and by 24 hours almost all CD20 had been lost consistent with a process previously described as shaving, during which R bound CD20 is pulled of the cell surface. Our data show that infusion of R induced a characteristic gene expression signature in CLL cells that is dominated by IFN response genes, many of which have well characterized pro-apoptotic functions. We conclude that signaling for apoptosis is less of a direct effect of rituximab and more due to a complex immune response to the rituximab coated CLL cells. From these data we hypothesize that strategies to diminish CD20 loss hold promise for improved efficacy of rituximab. We have tested a strategy of frequent low dose administration of rituximab. In this pilot study (ClinicalTrials.gov Identifier: NCT00366418) we investigated the feasibility of giving 20 mg rituximab subcutaneously thrice weekly for up to 12 weeks in 4 previously treated CLL patients. Subcutaneous rituximab was well-tolerated with minimal injection site reactions;a variable degree of efficacy was observed, likely influenced by the size of the patients B cell/CD20 burden. Subcutaneous RTX largely preserved CD20 expression on leukemic cells but the most effective therapeutic dosing regimen needs to be established. Lenalidomide is an immune modulatory drug shwoing promising phase II results. The mechanism of action and predictors of treatment outcome for this drug in CLL have not been defined. We initiated a phase II trial with a strong translation component to investigate the mechanism of action of lenalidomide in CLL. In CLL lenalidomide causes striking immune activation possibly leading to clearance of tumor cells. Patients with relapsed CLL were treated with lenalidomide and correlative studies assessed expression of costimulatory molecules on tumor cells, T-cell activation, cytokine levels, and changes in lymphocyte subsets. We found that lenalidomide upregulated the costimulatory molecule CD80 on CLL and Mantle Cell Lymphoma (MCL) cells but not on normal peripheral blood B cells in vitro. T-cell activation was apparent in CLL, weak in MCL, but absent in normal PBMCs and correlated with the upregulation of CD80 on B-cells. Strong CD80 upregulation and T-cell activation predicted more severe side effects, manifesting in 83% of patients as a cytokine release syndrome within 8-72 hours after the first dose. Serum levels of various cytokines, including TNF, increased during treatment. CD80 upregulation on tumor cells correlated with rapid clearance of leukemic cells from the peripheral blood. In contrast, neither the severity of the cytokine release syndrome, nor the degree of T-cell activation in vitro correlated with clinical response. We conclude that upregulation of CD80 on tumor cells and T-cell activation correlates with unique toxicities of lenalidomide in CLL. However, T-cell activation appeared to be dispensable for anti-tumor effects. In an extension of this study we are now using gene expression profiling to monitor treatment induced changes in the leukemic cells in blood and lymph nodes.